A model for the activation of plasma membrane calcium pump isoform 4b by calmodulin

Overexpression of the plasma membrane calcium pump (PMCA) isoform 4b by means of the baculovirus system enabled us, for the first time, to study the kinetics of calmodulin binding to this pump. This was done by stopped-flow fluorescence measurements using 2-chloro-(amino-Lys(75))-[6-[4-(N,N-diethylamino)phenyl]-1,3,5-triazin-4-yl]calmodulin (TA-calmodulin). Upon mixing with PMCA, the fluorescence of TA-calmodulin changed along a biphasic curve: a rapid and small increase in fluorescence was followed by a slow and large decrease that lasted about 100 s. The experiment was done at several PMCA concentrations. Global fitting nonlinear regression analysis of these results led to a model in which PMCA is present in two forms: a closed conformation and an open conformation. Calmodulin reacts with both conformations but reacts faster and with higher affinity for the open conformation. Measurements of the ATPase activity of PMCA under similar conditions revealed that the open form has higher ATPase activity than the closed one. Contrasting with the reaction with the whole pump, TA-calmodulin reacted rapidly (in about 2 s) with a calmodulin-binding peptide made after the sequence of the calmodulin-binding domain of PMCA (C28). Results of TA-calmodulin binding to C28 are explained by a simpler model, in which only an open conformation exists.     

Alternative Pathways for Association and Dissociation of the Calmodulin-binding Domain of Plasma Membrane Ca2+-ATPase Isoform 4b (PMCA4b)

The calmodulin (CaM)-binding domain of isoform 4b of the plasma membrane Ca(2+) -ATPase (PMCA) pump is represented by peptide C28. CaM binds to either PMCA or C28 by a mechanism in which the primary anchor residue Trp-1093 binds to the C-terminal lobe of the extended CaM molecule, followed by collapse of CaM with the N-terminal lobe binding to the secondary anchor Phe-1110 (Juranic, N., Atanasova, E., Filoteo, A. G., Macura, S., Prendergast, F. G., Penniston, J. T., and Strehler, E. E. (2010) J. Biol. Chem. 285, 4015-4024). This is a relatively rapid reaction, with an apparent half-time of ～1 s. The dissociation of CaM from PMCA4b or C28 is much slower, with an overall half-time of ～10 min. Using targeted molecular dynamics, we now show that dissociation of Ca(2+)-CaM from C28 may occur by a pathway in which Trp-1093, although deeply embedded in a pocket in the C-terminal lobe of CaM, leaves first. The dissociation begins by relatively rapid release of Trp-1093, followed by very slow release of Phe-1110, removal of C28, and return of CaM to its conformation in the free state. Fluorescence measurements and molecular dynamics calculations concur in showing that this alternative path of release of the PMCA4b CaM-binding domain is quite different from that of binding. The intermediate of dissociation with exposed Trp-1093 has a long lifetime (minutes) and may keep the PMCA primed for activation.     

Mechanism of calmodulin recognition of the binding domain of isoform 1b of the plasma membrane Ca(2+)-ATPase: kinetic pathway and effects of methionine oxidation

Calmodulin (CaM) binds to a domain near the C-terminus of the plasma membrane Ca2+-ATPase (PMCA), causing the release of this domain and relief of its autoinhibitory function. We investigated the kinetics of dissociation and binding of Ca2+-CaM with a 28-residue peptide [C28W(1b)] corresponding to the CaM-binding domain of isoform 1b of PMCA. CaM was labeled with a fluorescent probe on either the N-terminal domain at residue 34 or the C-terminal domain at residue 110. Formation of complexes of CaM with C28W(1b) results in a decrease in the fluorescence yield of the fluorophore, allowing the kinetics of dissociation or binding to be detected. Using a maximum entropy method, we determined the minimum number and magnitudes of rate constants required to fit the data. Comparison of the fluorescence changes for CaM labeled on the C-terminal or N-terminal domain suggests sequential and ordered binding of the C-terminal and N-terminal domains of CaM with C28W(1b). For dissociation of C28W(1b) from CaM labeled on the N-terminal domain, we observed three time constants, indicating the presence of two intermediate states in the dissociation pathway. However, for CaM labeled on the C-terminal domain, we observed only two time constants, suggesting that the fluorescence label on the C-terminal domain was not sensitive to one of the kinetic steps. The results were modeled by a kinetic mechanism in which an initial complex forms upon binding of the C-terminal domain of CaM to C28W(1b), followed by binding of the N-terminal domain, and then formation of a tight binding complex. Oxidation of methionine residues in CaM resulted in significant perturbations to the binding kinetics. The rate of formation of a tight binding complex was reduced, consistent with the poorer effectiveness of oxidized CaM in activating the Ca2+ pump.     

The plasma membrane Ca2+ pump isoform 4a differs from isoform 4b in the mechanism of calmodulin binding and activation kinetics: implications for Ca2+ signaling

The inhibition by the regulatory domain and the interaction with calmodulin (CaM) vary among plasma membrane calcium pump (PMCA) isoforms. To explore these differences, the kinetics of CaM effects on PMCA4a were investigated and compared with those of PMCA4b. The maximal apparent rate constant for CaM activation of PMCA4a was almost twice that for PMCA4b, whereas the rates of activation for both isoforms showed similar dependence on Ca2+. The inactivation of PMCA4a by CaM removal was also faster than for PMCA4b, and Ca2+ showed a much smaller effect (2- versus 30-fold modification). The rate constants of the individual steps that determine the overall rates were obtained from stopped-flow experiments in which binding of TA-CaM was observed by changes in its fluorescence. TA-CaM binds to two conformations of PMCA4a, an "open" conformation with high activity, and a "closed" one with lower activity. Compared with PMCA4b (Penheiter, A. R., Bajzer, Z., Filoteo, A. G., Thorogate, R., T?r?k, K., and Caride, A. J. (2003) Biochemistry 41, 12115-12124), the model for PMCA4a predicts less inhibition in the closed form and a much faster equilibrium between the open and closed forms. Based on the available kinetic parameters, we determined the constants to fit the shape of a Ca2+ signal in PMCA4b-overexpressing Chinese hamster ovary cells. Using the constants for PMCA4a, and allowing small variations in parameters of other systems contributing to a Ca2+ signal, we then simulated the effect of PMCA4a on the shape of a Ca2+ signal in Chinese hamster ovary cells. The results reproduce the published data (Brini, M., Coletto, L., Pierobon, N., Kraev, N., Guerini, D., and Carafoli, E. (2003) J. Biol. Chem. 278, 24500-24508), and thereby demonstrate the importance of altered regulatory kinetics for the different functional properties of PMCA isoforms.     

Biophysical characterization of the interaction of p21 with calmodulin: a mechanistic study

p21 is a protein with important roles in cell proliferation, cell cycle regulation and apoptosis. Several studies have demonstrated that its intracellular localization plays an important role in the functional regulation and binding of calmodulin favors its nuclear translocation. However, the detail mechanism of the interaction with p21 and calmodulin is not well understood. In this report, peptides derived from the C-terminal of p21 that cover the binding domain of calmodulin were used to investigate the association of p21 with calmodulin. We found p21(141-164) interaction with Ca(2+)-saturated dansyl-labelled calmodulin caused a significant increase in dansyl fluorescence intensity and a blue shift of the maximum emission from 510 to 475 nm. The Trp fluorescence intensities of mutated p21(141-164) peptides (F150W, Y151W and F159W) increased upon binding to Ca(2+)-saturated calmodulin and fluorescence maxima were blue shifted from 350 nm to 330 nm. The results suggested p21(141-164) is most likely buried in the hydrophobic binding tunnel of calmodulin. Both dansyl and Trp fluorescence titrations generated dissociation constants around 0.1 muM and a stoichiometry of 1:1, which was further confirmed by nondenaturing gel band shift electrophoresis. Fluorescence titrations and Trp fluorescence quenching results indicated electrostatic interaction is involved in this association. Upon binding to calmodulin, p21(141-164) remained largely unstructured and showed only about 15% alpha-helix. In contrast to other calmodulin binding peptide, the dominant force in the association of p21(141-164) with calmodulin may be electrostatic interaction. Our results would be helpful for understanding the molecular details of p21 and calmodulin interaction.     

Binding of calcium by calmodulin: influence of the calmodulin binding domain of the plasma membrane calcium pump.

The interaction between calmodulin and synthetic peptides corresponding to the calmodulin binding domain of the plasma membrane Ca2+ pump has been studied by measuring Ca2+ binding to calmodulin. The largest peptide (C28W) corresponding to the complete 28 amino acid calmodulin binding domain enhanced the Ca2+ affinity of calmodulin by more than 100 times, implying that the binding of Ca2+ increased the affinity of calmodulin for the peptide by more than 10(8) times. Deletion of the 8 C-terminal residues from peptide C28W did not decrease the affinity of Ca2+ for the high-affinity sites of calmodulin, but it decreased that for the low-affinity sites. A larger deletion (13 residues) decreased the affinity of Ca2+ for the high-affinity sites as well. The data suggest that the middle portion of peptide C28W interacts with the C-terminal half of calmodulin. Addition of the peptides to a mixture of tryptic fragments corresponding to the N- and C-terminal halves of calmodulin produced a biphasic Ca2+ binding curve, and the effect of peptides was different from that on calmodulin. The result shows that one molecule of peptide C28W binds both calmodulin fragments. Interaction of the two domains of calmodulin through the central helix is necessary for the high-affinity binding of four Ca2+ molecules.     

Response of GWALP transmembrane peptides to changes in the tryptophan anchor positions

While the interfacial partitioning of charged or aromatic anchor residues may determine the preferred orientations of transmembrane peptide helices, the dependence of helix orientation on anchor residue position is not well understood. When anchor residue locations are changed systematically, some adaptations of the peptide-lipid interactions may be required to compensate for the altered interfacial interactions. Recently, we have developed a novel transmembrane peptide, termed GW(5,19)ALP23 (acetyl-GGALW(5)LALALALALALALW(19)LAGA-ethanolamide), which proves to be a well-behaved sequence for an orderly investigation of protein-lipid interactions. Its roughly symmetric nature allows for shifting the anchoring Trp residues by one Leu-Ala pair inward (GW(7,17)ALP23) or outward (GW(3,21)ALP23), thus providing fine adjustments of the formal distance between the tryptophan residues. With no other obvious anchoring features present, we postulate that the inter-Trp distance may be crucial for aspects of the peptide-lipid interaction. Importantly, the amino acid composition is identical for each of the resulting related GWALP23 sequences, and the radial separation between the pairs of Trp residues on each side of the transmembrane α-helix remains similar. Here we address the adaptation of the aforementioned peptides to the varying Trp locations by means of solid-state (2)H nuclear magnetic resonance experiments in varying lipid bilayer membrane environments. All of the GW(x,y)ALP23 sequence isomers adopt transmembrane orientations in DOPC, DMPC, and DLPC environments, even when the Trp residues are quite closely spaced, in GW(7,17)ALP23. Furthermore, the dynamics for each peptide isomer are less extensive than for peptides possessing additional interfacial Trp residues. The helical secondary structure is maintained more strongly within the Trp-flanked core region than outside of the Trp boundaries. Deuterium-labeled tryptophan indole rings in the GW(x,y)ALP23 peptides provide additional insights into the behavior of the Trp side chains. A Trp side chain near the C-terminus adopts a different orientation and undergoes somewhat faster dynamics than a corresponding Trp side chain located an equivalent distance from the N-terminus. In contrast, as the inter-Trp distance changes, the variations among the average orientations of the Trp indole rings at either terminus are systematic yet fairly small. We conclude that subtle adjustments to the peptide tilt, and to the N- and C-terminal Trp side chain torsion angles, permit the GW(x,y)ALP23 peptides to maintain preferred transmembrane orientations while adapting to lipid bilayers with differing hydrophobic thicknesses.     

Substitution of the N-terminal segment of the plasma membrane Ca pump isoform 4 by that of isoform 1 results in a fully functional chimeric enzyme.

The N-terminal segment of the plasma membrane Ca2+ pump (PMCA) is one of the most variable regions among the four isoforms of the enzyme and its functional importance is unknown. In the present work, the N-terminal segment of the highly active C-terminally truncated h4 mutant, h4(ct120) was modified either by substituting residues 18-43 by residues 43-75 or by replacing residues 1-75 by the homologous region from isoform h1 (residues 1-79). Immunoblot analysis of microsomal membranes from transfected COS-1 cells showed that the two N-terminally mutated proteins were correctly expressed at a level similar to that of h4(ct120). Measurements of the Ca2+ uptake by microsomal vesicles from transfected COS-1 cells indicated that mutant (18-43-->43-75)h4(ct120) had only negligible Ca2+ transport activity while the chimeric (n1-79)h1h4(ct120) enzyme was fully capable of functioning as a calcium pump.Like h4(ct120), the chimeric mutant was not stimulated further by calmodulin, and was inhibited to a similar degree by the C28R2 peptide corresponding to the calmodulin binding autoinhibitory region of the pump. Moreover, the apparent affinity for Ca2+ and the ATP dependence of the chimeric enzyme were similar to those of the h4(ct120) pump suggesting that the variability of sequence between the N-terminal segment of PMCA isoforms h1 and h4 involves amino acid substitutions that do not substantially change the behavior of the h4 enzyme. Altogether, these results demonstrate that for activity the h4 Ca pump requires a specific amino acid sequence at its N-terminus, and the essential elements for a fully active enzyme can be provided by the N-terminal segment of isoform h1 despite the variability.     

Calmodulin binding properties of peptide analogues and fragments of the calmodulin-binding domain of simian immunodeficiency virus transmembrane glycoprotein 41

The calcium-regulatory protein calmodulin (CaM) can bind with high affinity to a region in the cytoplasmic C-terminal tail of glycoprotein 41 of simian immunodeficiency virus (SIV). The amino acid sequence of this region is (1)DLWETLRRGGRW(13)ILAIPRRIRQGLELT(28)L. In this work, we have used near- and far-uv CD, and fluorescence spectroscopy, to study the orientation of this peptide with respect to CaM. We have also studied biosynthetically carbon-13 methyl-Met calmodulin by (1)H, (13)C heteronuclear multiple quantum coherence NMR spectroscopy. Two Trp-substituted peptides, SIV-W3F and SIV-W12F, were utilized in addition to the intact SIV peptide. Two half-peptides, SIV-N (residues 1-13) and SIV-C (residues 13-28) were also synthesized and studied. The spectroscopic results obtained with the SIV-W3F and SIV-W12F peptides were generally consistent with those obtained for the native SIV peptide. Like the native peptide, these two analogues bind with an alpha-helical structure as shown by CD spectroscopy. Fluorescence intermolecular quenching studies suggested binding of Trp3 to the C-lobe of CaM. Our NMR results show that SIV-N can bind to both lobes of calcium-CaM, and that it strongly favors binding to the C-terminal hydrophobic region of CaM. The SIV-C peptide binds with relatively low affinity to both halves of the protein. These data reveal that the intact SIV peptide binds with its N-terminal region to the carboxy-terminal region of CaM, and this interaction initiates the binding of the peptide. This orientation is similar to that of most other CaM-binding domains.     

NMR conformational analyses on (des-bromo) neuropeptide B [1-23] and neuropeptide W [1-23]: the importance of alpha-helices, a cation-pi interaction and a beta-turn

The preferred conformations of the orphan G-protein coupled receptor agonists (des-bromo) neuropeptide B [1-23] and neuropeptide W [1-23], referred to as NPB and NPW, have been determined by (1)H NMR, CD, and molecular modeling. The sequences of NPB and NPW are WYKPAAGHSSYSVGRAAGLLSGL and WYKHVASPRYHTVGRAAGLLMGL, respectively. These are hypothalamic peptides that exert their biological actions on GPR7 and GPR8 receptors. Micellar solutions using the membrane mimetic, sodium dodecylsulphate-d(25) (SDS), were used to mimic a physiological environment for the peptides. The secondary structure of NPB consists of a type II beta-turn involving residues Lys(3) to Ala(6). The C-terminal region of NPB exists in a conformational equilibrium between different secondary structures, including an alpha-helix from residues Arg(15) to Ser(21), and a 3(10)-helix from residues Ser(12) to Ser(21). The N-terminus of NPW exhibits a cation-pi interaction between the Lys(3) side chain and the quadrupole moment of the Trp(1) indole group. At the C-terminus of NPW, a well-defined alpha-helical conformation exists from Arg(15) to Met(21). As NPB and NPW have 91% sequence homology from residues Val(13) to Leu(23), with only residue 21 differing between the two peptides, the similar C-terminal secondary structures of these two peptides are consistent with the sequences. This is supported by the similar CD spectra. The different secondary structures at the N-termini for NPB and NPW point to the importance of the N-terminus in receptor binding. This is consistent with the work of Fujii et al. [J. Biol. Chem. 277, 34010-34016 (2002)] who observed that iodination of the NPB Tyr(2) resulted in decreased agonistic activity at GPR7. In addition, Tanaka et al. [Proc. Natl. Acad. Sci. USA 100, 6251-6256 (2003)] showed that deletion of Trp(1) from NPB or NPW drastically decreased activity at GPR7 for NPB and GPR7 and GPR8 for NPW. Therefore, we postulate that the N-terminus is involved in membrane recognition and receptor binding.     

Binding of simple peptides, hormones, and neurotransmitters by calmodulin

We have prepared a fluorescent conjugate of porcine calmodulin with 5-(dimethylamino)-1-naphthalene-sulfonyl chloride that is highly sensitive to both calcium binding and protein binding. We have used the fluorescence of this conjugate in addition to the intrinsic peptide fluorescence to show that adrenocorticotropic hormone (ACTH), beta-endorphin, glucagon, and substance P undergo calcium-dependent binding by calmodulin, with competition for common binding sites. The dissociation constants determined in the presence of 0.85 mM CaCl2 and 0.2 N KC1, pH 7.3 at 25 degrees C, range from 1.5 muM to 3.4 muM. The alpha-melanocyte-stimulating hormone, bombesin, and somatostatin also bind, with dissociation constants between 60 muM and 90 muM. Angiotensins I and III, bradykinin, neurotensin, physalaemin, substance P octapeptide, insulin, and Leu- and Met-enkephalin show little or no binding. Sequence comparisons show that the peptides that bind calmodulin well contain regions structurally similar to the recognition sequence for the cAMP-dependent protein kinase and to the sequences surrounding phosphorylated serine residues in several calmodulin binding proteins. This result suggests that modification of calmodulin binding sites in calmodulin-dependent proteins is one of the functions of protein kinase. Calcium has a dual role in peptide binding by calmodulin. The occupation of calcium binding sites having a pK approximately 4 results in a 2-fold increase in peptide binding affinity.     

Mediating molecular recognition by methionine oxidation: conformational switching by oxidation of methionine in the carboxyl-terminal domain of calmodulin

The C-terminus of calmodulin (CaM) functions as a sensor of oxidative stress, with oxidation of methionine 144 and 145 inducing a nonproductive association of the oxidized CaM with the plasma membrane Ca(2+)-ATPase (PMCA) and other target proteins to downregulate cellular metabolism. To better understand the structural underpinnings and mechanism of this switch, we have engineered a CaM mutant (CaM-L7) that permits the site-specific oxidation of M144 and M145, and we have used NMR spectroscopy to identify structural changes in CaM and CaM-L7 and changes in the interactions between CaM-L7 and the CaM-binding sequence of the PMCA (C28W) due to methionine oxidation. In CaM and CaM-L7, methionine oxidation results in nominal secondary structural changes, but chemical shift changes and line broadening in NMR spectra indicate significant tertiary structural changes. For CaM-L7 bound to C28W, main chain and side chain chemical shift perturbations indicate that oxidation of M144 and M145 leads to large tertiary structural changes in the C-terminal hydrophobic pocket involving residues that comprise the interface with C28W. Smaller changes in the N-terminal domain also involving residues that interact with C28W are observed, as are changes in the central linker region. At the C-terminal helix, (1)H(alpha), (13)C(alpha), and (13)CO chemical shift changes indicate decreased helical character, with a complete loss of helicity for M144 and M145. Using (13)C-filtered, (13)C-edited NMR experiments, dramatic changes in intermolecular contacts between residues in the C-terminal domain of CaM-L7 and C28W accompany oxidation of M144 and M145, with an essentially complete loss of contacts between C28W and M144 and M145. We propose that the inability of CaM to fully activate the PMCA after methionine oxidation originates in a reduced helical propensity for M144 and M145, and results primarily from a global rearrangement of the tertiary structure of the C-terminal globular domain that substantially alters the interaction of this domain with the PMCA.     

Fluorescence of native single-Trp mutants in the lactose permease from Escherichia coli: structural properties and evidence for a substrate-induced conformational change.

Six single-Trp mutants were engineered by individually reintroducing each of the native Trp residues into a functional lactose permease mutant devoid of Trp (Trp-less permease; Menezes ME, Roepe PD, Kaback HR, 1990, Proc Natl Acad Sci USA 87:1638-1642), and fluorescent properties were studied with respect to solvent accessibility, as well as alterations produced by ligand binding. The emission of Trp 33, Trp 78, Trp 171, and Trp 233 is strongly quenched by both acrylamide and iodide, whereas Trp 151 and Trp 10 display a decrease in fluorescence in the presence of acrylamide only and no quenching by iodide. Of the six single-Trp mutants, only Trp 33 exhibits a significant change in fluorescence (ca. 30% enhancement) in the presence of the substrate analog beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG). This effect was further characterized by site-directed fluorescent studies with purified single-Cys W33-->C permease labeled with 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS). Titration of the change in the fluorescence spectrum reveals a 30% enhancement accompanied with a 5-nm blue shift in the emission maximum, and single exponential behavior with an apparent KD of 71 microM. The effect of substrate binding on the rate of MIANS labeling of single-Cys 33 permease was measured in addition to iodide and acrylamide quenching of the MIANS-labeled protein. Complete blockade of labeling is observed in the presence of TDG, as well as a 30% decrease in accessibility to iodide with no change in acrylamide quenching. Overall, the findings are consistent with the proposal (Wu J, Frillingos S, Kaback HR, 1995a, Biochemistry 34:8257-8263) that ligand binding induces a conformational change at the C-terminus of helix I such that Pro 28 and Pro 31, which are on one face, become more accessible to solvent, whereas Trp 33, which is on the opposite face, becomes less accessible to the aqueous phase. The findings regarding accessibility to collisional quenchers are also consistent with the predicted topology of the six native Trp residues in the permease.     

The interaction of calmodulin with amphiphilic peptides

Calmodulin has recently been shown to form exceptionally tight, calcium-dependent complexes with several natural peptides (Kdiss greater than 10(-7) M). These peptides were demonstrated to be capable of forming basic, amphiphilic alpha-helices. To further illustrate the importance of this structural feature for calmodulin binding, several other amphiphilic alpha-helical peptides were tested for their ability to bind calmodulin. To monitor complexes of high affinity (greater than 10(8) M-1), a new competition assay was devised with Sepharose 4B-conjugated melittin. Stoichiometries were assessed by electrophoresis and equilibrium size exclusion chromatography. Three peptides, which were designed to form idealized amphiphilic alpha-helices were tested. The basic peptides, N alpha-9-fluorenylmethoxycarboxyl-(FMOC)-(Leu-Lys-Lys-Leu-Leu-Lys-L eu)1 and FMOC-(Leu-Lys-Lys-Leu-Leu-Lys-Leu)2 bind calmodulin in a 1:1 complex with dissociation constants of 150 and 3 nM, respectively. The acidic peptide, FMOC-(Leu-Glu-Glu-Leu-Leu-Glu-Leu)2 failed to bind calmodulin, even at micromolar concentrations. Complex formation between calmodulin and the 14-residue basic peptide leads to an increase in the helicity of the complex which is attributed to an increase of about 50% in the helicity of the peptide. Calmodulin also interacts with the neutral alpha-helical peptide toxin delta-hemolysin. Concomitant with binding, the fluorescence maximum of the unique Trp residue increases 2-fold and is blue-shifted. A dissociation constant could not be unambiguously estimated though, since delta-hemolysin has a strong tendency to self-aggregate. The above data support our hypothesis that a basic, amphiphilic alpha-helix is a structural feature which underlies the calmodulin-binding properties common to a variety of peptides.     

Site-directed mutagenesis of conserved C-terminal tyrosine and tryptophan residues of PsbO, the photosystem II manganese-stabilizing protein, alters its activity and fluorescence properties

The extrinsic photosystem II PsbO subunit (manganese-stabilizing protein) contains near-UV CD signals from its complement of aromatic amino acid residues (one Trp, eight Tyr, and 13 Phe residues). Acidification, N-bromosuccinimide modification of Trp, reduction or elimination of a disulfide bond, or deletion of C-terminal amino acids abolishes these signals. Site-directed mutations that substitute Phe for Trp241 and Tyr242, near the C-terminus of PsbO, were used to examine the contribution of these residues to the activity and spectral properties of the protein. Although this substitution is, in theory, conservative, neither mutant binds efficiently to PSII, even though these proteins appear to retain wild-type solution structures. Removal of six residues from the N-terminus of the W241F mutant restores activity to near-wild-type levels. The near-UV CD spectra of the mutants are modified; well-defined Tyr and Trp peaks are lost. Characterizations of the fluorescence spectra of the full-length WF and YF mutants indicate that Y242 contributes significantly to PsbO's Tyr fluorescence emission and that an excited-state tyrosinate could be present in PsbO. Deletion of W241 shows that this residue is a major contributor to PsbO's fluorescence emission. Loss of function is consistent with the proposal that a native C-terminal domain is required for PsbO binding and activity, and restoration of activity by deletion of N-terminal amino acids may provide some insights into the evolution of this important photosynthetic protein.     

Photoaffinity labeling study of the interaction of calmodulin with the plasma membrane Ca2+ pump.

Bovine brain calmodulin was labeled with synthetic peptides corresponding to the calmodulin-binding domain of the erythrocyte plasma membrane Ca(2+)-ATPase. One 20-amino acid peptide and two 28-amino acid peptides were used, carrying L-4'-(1-azi-2,2,2-trifluoroethyl)phenylalanine residues in position 9 (peptides C20W* and C28W*) and position 25 (peptide C28WC*), respectively. The localization of the contact regions between calmodulin and the N- and C-terminal portions of the peptides was the aim of this study. The three peptides were N-terminally blocked with a 3H-labeled acetyl group to facilitate the identification of labeled fragments after isolation and digestion. The binding site for phenylalanine 25 was identified in the N-terminal domain of calmodulin while the phenylalanine derivative in position 9 labeled the C-terminal domain. Fluorescence studies using the dansylated N- and C-terminal halves of calmodulin and peptide C20W corresponding to the first 20 amino acids of the calmodulin-binding domain showed that only the C-terminal lobe of calmodulin had high affinity for the peptide (KD in the nanomolar range).     

Target recognition by calmodulin: dissecting the kinetics and affinity of interaction using short peptide sequences.

The interaction between calmodulin (CaM) and peptide M13, its target binding sequence from skeletal muscle myosin light chain kinase, involves predominantly two sets of interactions, between the N-terminal target residues and the C-domain of calmodulin, and between the C-terminal target residues and the N-domain of calmodulin (Ikura M et al., 1992, Science 256:632-638). Using short synthetic peptides based on the two halves of the target sequence, the interactions with calmodulin and its separate C-domain have been studied by fluorescence and CD spectroscopy, calcium binding, and kinetic techniques. Peptide WF10 (residues 1-10 of M13) binds to CaM with Kd approximately 1 microM; peptide FW10 (residues 9-18 of M13, with Phe-17-->Trp substitution) binds to CaM with Kd approximately 100 microM. The effect of peptide WF10 on calcium binding to calmodulin produces a biphasic saturation curve, with marked enhancement of affinity for the binding of two calcium ions to the C-domain, forming a stable half-saturated complex, Ca2-CaM-peptide, and confirming the functional importance of the interaction of this sequence with the C-domain. Stopped-flow studies show that the EGTA-induced dissociation of WF10 from Ca4-CaM proceeds by a reversible relaxation mechanism from a kinetic intermediate state, also involving half-saturation of CaM, and the same mechanism is evident for the full target peptide. Interaction of the N-terminal target residues with the C-domain is energetically the most important component, but interaction of calmodulin with the whole target sequence is necessary to induce the full cooperative interaction of the two contiguous elements of the target sequence with both N- and C-domains of calmodulin. Thus, the interaction of calmodulin with the M13 sequence can be dissected on both a structural and kinetic basis into partial reactions involving intermediates comprising distinct regions of the target sequence. We propose a general mechanism for the calcium regulation of calmodulin-dependent enzyme activation, involving an intermediate complex formed by interaction of the calmodulin C-domain and the corresponding part of the target sequence. This intermediate species can function to regulate the overall calcium sensitivity of activation and to determine the affinity of the calmodulin target interaction.     

Role of strong anchor residues in the effective binding of 10-mer and 11-mer peptides to HLA-A*2402 molecules

The binding capacity of one-hundred-and-seventy-two 8-mer to 11-mer peptides carrying HLA-A24 anchor residues to HLA-A^*2402 molecules was analyzed by using a HLA class I stabilization assay. Most (76.2%) of these peptides bound to HLA-A^*2402 molecules. These results confirmed previous findings that Tyr and Phe at P2 as well as Phe, Trp, Ile, and Leu at the C-terminus were main anchor residues for HLA-A^*2402. Tyr at P2 was a stronger anchor residue than Phe, while bulky aromatic hydrophobic residues Phe and Trp at the C-terminus are stronger anchors than aliphatic hydrophobic residues Ile and Leu. These results were also supported by an analysis using a panel of mutated 9-mer peptides at P2 and P9. Taken together, these results suggest that HLA-A^*2402 molecules have deep B- and F-pockets because they favor peptides carrying bulky aromatic hydrophobic residues at P2 and the C-terminus. The affinity of 8-mer peptides was significantly lower than that of 9-mer to 11-mer peptides, while there was no difference in affinity between 9-mer, 10-mer, and 11-mer peptides. The affinity of peptides carrying bulky aromatic hydrophobic residues at the C-terminus was higher than that of peptides carrying aliphatic hydrophobic residues in each of the 8-mer to 11-mer peptides, though the greatest difference in affinity was observed in 11-mer peptides. The strong interaction of side chains of these anchor residues with the corresponding pockets may permit the effective binding of 10-mer and 11-mer peptides to HLA-A^*2402 molecules.     

Effects of volatile anesthetic on the Ca2+-ATPase activation by dimerization. Distance-dependent quenching analysis and fluorescence energy transfer studies.

The phenomenological distance-dependent quenching (DDQ) model was employed to investigate the character of the interaction between volatile anesthetics (VAs) and the plasma membrane Ca2+-ATPase (PMCA). The simultaneous analysis of the frequency-domain and steady-state data of tryptophan (Trp) fluorescence quenching by a VA points to a specific character of the apparent quenching effect of the VA, possibly arising from a significant contribution of static quenching. The apparent contributions of both static and dynamic quenching may be due to VA binding in the PMCA, which results in the modification of the conformational substates of the enzyme. To characterize further the molecular consequences of VA binding, we investigated its effects on the process of PMCA activation by self-association. VA shifted the equilibrium from enzyme dimers to monomers, as monitored by the loss of fluorescence energy transfer. The shift was apparently due to the VA-induced decrease in the affinity of PMCA molecules for self-association. Addition of a large molecular mass dextran to increase the proximity between enzyme monomers induced re-association of the VA-impaired PMCA, while the Ca2+-ATPase activity was not recovered. The results are congruent with a dual VA effect on PMCA, a shift in the monomer/dimer equilibrium, and an inactivation of both monomers and dimers.